The stability of proteins used in medical treatment is an important concern in medicine. Protein degradation can be classified as physical or chemical degradation. Physical degradation is caused by a change in conformation that leads to aggregation of the protein and formation of protein fibrils. Chemical degradation of proteins entails a change in the pattern of covalent bonds between atoms, such as breakage or interchange of disulfide bridges, deamination or transamination of the protein.
Administration of insulin has long been established as a treatment for diabetes mellitus. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A-chain, containing 21 residues, and a B-chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B-chain (residue B30) to the N-terminal residue of the A-chain (FIG. 1A). Although the structure of proinsulin has not been determined, a variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide (FIG. 1B). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIG. 1B) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). Assembly and disassembly of native oligomers is thus intrinsic to the pathway of insulin biosynthesis, storage, secretion, and action.
Insulin readily misfolds in vitro to form a prototypical amyloid. Unrelated to native assembly, fibrillation is believed to occur via an amyloidogenic partial fold (FIG. 1C). Factors that accelerate or hinder fibrillation have been extensively investigated in relation to pharmaceutical formulations. Zinc-free insulin is susceptible to fibrillation under a broad range of conditions and is promoted by factors that impair native dimerization and higher-order self-assembly. A storage form of insulin in the pancreatic β-cell and in the majority of pharmaceutical formulations is believed to be stabilized by axial zinc ions coordinated by the side chains of insulin amino acids, specifically the HisB10 residues. Formulation of insulin or insulin analogues as a zinc-stabilized hexamer retards but does not prevent fibrillation, especially above room temperature and on agitation.
Amino-acid substitutions in the A- and/or B-chains of insulin have widely been investigated for possible favorable effects on the pharmacokinetics of insulin action following subcutaneous injection. Examples are known in the art of substitutions that accelerate or delay the time course of absorption. The former analogues collectively define the “meal-time” insulin analogues because patients with diabetes mellitus may inject such rapid-acting formulations at the time of a meal whereas the delayed absorption of wild-type human insulin or animal insulins (such as porcine insulin or bovine insulin) makes it necessary to inject these formulations 30-45 minutes prior to a meal. The substitutions are designed to destabilize the zinc insulin hexamer by altering the steric or electrostatic complementarity of subunit interfaces and thereby to facilitate the rapid dissociation of the zinc insulin hexamer after subcutaneous administration. A side consequence of this design is decreased chemical and physical stability of the insulin analogue formulation, which can limit the shelf life of the formulation at room temperature and cause occlusion of external insulin pumps due to formation of insulin fibrils. The present invention concerns the prevention of this increased susceptibility to fibrillation and chemical degradation by meal-time insulin analogues. In addition to patient convenience and quality of life, the rapid-acting insulin analogues may increase the safety of intensive multiple-injection regimens intended to achieve tight glycemic control. Rapid absorption is also reported to enhance the efficacy and safety of glycemic control obtained by the subcutaneous injection of an insulin analogue formulation by an external insulin pump. Despite these favorable properties, such substitutions (such as AspB28 in Novalog® and [LysB28, ProB29] in Humalog®) can be and often are associated with more rapid fibrillation and poorer physical stability. Similar decreases in chemical and physical stability are expected for Apidra® (insulin glulisine, which contains the amino-acid substitutions AsnB3→Lys and LysB29→Glu).
The decreased chemical stability of the meal-time insulin analogues and their increased susceptibility to fibrillation appears to be intrinsic to their design. Since substitutions were introduced to destabilize the hexamer and therefore make the smaller monomer more available for rapid absorption, a higher proportion of the susceptible monomer will necessarily be present at equilibrium than would be the case with wild-type insulin. It has previously not been clear how this intrinsic problem could be overcome to restore a wild-type level of stability and resistance to fibrillation. Indeed, a series of ten analogues of human insulin, including AspB28-insulin and AspB10-insulin has been tested for susceptibility to fibrillation. All ten were found to be more susceptible to fibrillation at pH 7.4 and 37° C. than is human insulin. The ten substitutions were located at diverse sites in the insulin molecule and are likely to be associated with a wide variation of changes in classical thermodynamic stability. These results suggest that substitutions that protect an insulin analogue from fibrillation under pharmaceutical conditions are rare; no structural criteria or rules are apparent for their design. The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. It is therefore not obvious from theory whether a given amino-acid substitution in the insulin molecule will increase or decrease the risk of fibrillation in accord with the experimental experience cited above. While it is possible for a particular amino-acid substitution to simultaneously enhance the thermodynamic stability of insulin and its resistance to fibrillation, such substitutions have not been identified. Such concordance would be desirable in a clinical insulin analogue formulation.
Fibrillation, which is a serious concern in the manufacture, storage, and use of insulin and insulin analogues for diabetes treatment, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, diabetic individuals optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for diabetic patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus, and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable blood glucose level fluctuations or even dangerous hyperglycemia. At least one recent report has indicated that lispro insulin (an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; trade name Humalog®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters.
The major barrier to the storage and practical use of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 30° C. is accelerated fibrillation of the protein. The major reason for limitations to the shelf life of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 10° C. is due to fibrillation of the protein. As noted above, fibrillation is of special concern for fast-acting or “mealtime” insulin analogues (such as Humalog® and Novalog®), particularly when these formulations are diluted by the patient and stored at room temperature for more than 15 days.
Development of fibrillation-resistant insulin analogues would not in itself lead to proteins of indefinite shelf life due to eventual degradation of the protein by chemical modification. Whereas fibrillation represents a change in the structure and spatial relationships between insulin molecules by means of altered non-covalent interactions, chemical modification alters the pattern of covalent bonding between atoms in the insulin molecule. Examples of chemical degradation are breakage of disulfide bridges, formation of non-native disulfide bridges between insulin molecules to form covalent dimers and higher-order polymers, deamination of an asparagine side chain to form an aspartic-acid side chain, and rearrangement of aspartic acid to form iso-aspartic acid within the insulin molecule. Whereas the propensity of an insulin analogue to form fibrils is not correlated with its global thermodynamic stability, enhancing the thermodynamic stability of the insulin molecule has been established to protect the protein from chemical degradation. Therefore, among meal-time analogues, a desirable property would also be enhanced thermodynamic stability to confer simultaneous protection from chemical degradation. The combination of resistance to fibrillation and resistance to chemical degradation would potentially optimize the safe and effective use of an insulin analogue within the reservoir of an insulin pump and to extend the shelf life of an insulin analogue formulation at or above room temperature.
Amino-acid substitutions in insulin have been investigated for effects on thermodynamic stability and biological activity. No consistent relationship has been observed between stability and activity. Whereas some substitutions that enhance thermodynamic stability also enhance binding to the insulin receptor, other substitutions that enhance stability impede such binding. The effects of substitution of ThrA8 by several other amino acids has been investigated in wild-type human insulin and in the context of an engineered insulin monomer containing three unrelated substitutions in the B-chain (HisB10→Asp, ProB28→Lys, and LysB29→Pro) have been reported. Although a range of effects has been observed, no correlation exists between activity and thermodynamic stability. The substitutions HisA8 and ArgA8 have been reported to markedly augment the thermodynamic stability of wild-type human insulin, but the initial studies were conducted at high pH (pH 8.0) and in extremely low ionic strength (10 mM Tris/ClO4—); re-investigation of the HisA8 analog at pH 7.4 and moderate ionic strength (10 mM potassium phosphate buffer and 50 mM KCl) indicated much smaller effects. The substitution HisA8 has been reported to delay the fibrillation of wild-type insulin at pH 1.6-2.0 at 60° C. (conditions not relevant to the formulation or use of insulin analogues in the treatment of diabetes mellitus), but its effects at neutral pH and at lower temperatures have not been described. Comparative studies of the kinetics of fibrillation at pH 1.6-2.0 at 60° C. and at pH 7.4 at 37° C. have demonstrated that effects at acidic pH are uncorrelated with effects at neutral pH pertinent to pharmaceutical formulation. No data have been published describing effects of substitutions at A8 or elsewhere on the stability properties of meal-time insulin analogues.
Meal-time insulin analogues are ordinarily formulated as zinc insulin hexamers. Because the process of fibrillation proceeds via a conformationally altered insulin monomer, sequestration of the monomer within a native assembly reduces the concentration of the susceptible monomer. Such assembly also enhances thermodynamic stability, retarding chemical degradation. In addition, insulin assembly damps conformational fluctuations, reducing the rates of both physical and chemical degradation. Because of these advantages, a common method of formulation is to form zinc-stabilized insulin hexamers, the predominant form of the protein in the products Humalin™ (Eli Lilly and Co.), Humalog™ (Eli Lilly and Co.), Novalin™ (Novo-Nordisk), and Novalog™ (Novo-Nordisk). Analogous principles (but in the absence of zinc) pertain to the fast-acting analog Glulisine (Aprida™, manufactured by Sanofi-Aventis). Because of the decreased stability of meal-time insulin analogues, there is a need to identify amino-acid substitutions on the surface of the insulin analogue hexamer that restores or improves the chemical and physical stabilities of the analogue to levels similar to or exceeding those of wild-type human insulin. Such stabilizing substitutions would in principle be beneficial irrespective of whether the insulin analogue is formulated as a solution of soluble zinc-free hexamers, as a solution of soluble zinc hexamers, as a microcrystalline suspension or precipitate of protamine-stabilized zinc hexamers, including but not restricted to regular, NPH, lente, and semi-lente formulations or mixtures thereof.
The problem of chemical degradation and fibrillation is encountered not only among rapid-acting insulin analogues, but also in the formulation of long-acting insulin analogues under acidic conditions. Such analogues, exemplified but not restricted to [GlyA21, ArgB31, ArgB32]-insulin (insulin glargine or Lantus®), contain amino-acid substitutions and/or extensions of the A- or B-chains designed to shift the isoelectric point of the insulin analogue upward toward neutrality. The analogues are typically formulated as soluble insulin monomers, dimers, and higher-order oligomers at pH<5 under which conditions zinc-mediated assembly is impaired by protonation of HisB10. Prolonged absorption is achieved by aggregation and precipitation of the insulin analogue in the subcutaneous tissue due to a shift in pH toward 7.4. Under the acidic conditions of formulation, long-acting analogues of this class are susceptible to chemical and physical degradation due to the non-negligible concentration of unprotected monomer in the self-association equilibrium in solution. Therefore, there is a need for amino acid substitutions which stabilize long-acting insulin analogues under conditions of acidic formulation.